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Free-living amoebae belong to the genus Acanthamoeba; can feed on microbial population by phagocytosis, and with the capability to act as a reservoir and a vehicle of microorganisms to susceptible host. Therefore, the role of endosymbiosis in the pathogenesis of Acanthamoeba is complex and not fully understood. The aim of the present study was to identify bacterial, fungal, and human adenovirus (HADV) endosymbionts as well as evaluating the endosymbionts role of such organisms in the pathogenesis of Acanthamoeba in keratitis patients living in Iran. Fifteen Acanthamoeba (T4 genotype) isolates were recovered from corneal scrapes and contact lenses of patients with keratitis. Cloning and purification was performed for all isolate. Gram staining was performed to identify bacterial endosymbionts. DNA extraction, PCR, and nested PCR was set up to identify endosymbiont of amoeba. Evaluation of pathogenicity was conducted by osmo-tolerance and thermo-tolerance assays and cell culture, and then CPE (cytopathic effect) was survey. Statistical analysis was used between Acanthamoeba associated endosymbionts and Acanthamoeba without endosymbiont at 24, 48, 72, and 96 h. A p value < 0.05 was considered as significant, statistically. A total of 9 (60%) Acanthamoeba (T4 genotypes) isolates were successfully cloned for detecting microorganism endosymbionts. The only isolate negative for the presence of endosymbiont was ICS9. ICS7 (Pseudomonas aeruginosa , Aspergillus sp., and human adenovirus endosymbionts) and ICS2 (Escherichia coli endosymbiont) isolates were considered as Acanthamoeba associated endosymbionts. ICS7 and ICS2 isolates were highly pathogen whereas ICS9 isolate showed low pathogenicity in pathogenicity evaluated. Positive CPE for ICS7 and ICS2 isolates and negative CPE for ICS9 isolate were observed in cell culture. The average number of cells, trophozoites, and cysts among ICS7, ICS2, and ICS9 isolates at 24, 48, 72, and 96 h was significant. This is the first survey on microbial endosymbionts of Acanthamoeba in keratitis patients of Iran, and also the first report of Aspergillus sp, Achromobacter sp. , Microbacterium sp., Brevibacillus sp , Brevundimonas sp and Mastadenovirus sp in Acanthamoeba as endosymbionts. Our study demonstrated that microbial endosymbionts can affect the pathogenicity of Acanthamoeba ; however, further research is required to clarify the exact pattern of symbiosis, in order to modify treatment protocol. Image 1 • Our study demonstrated that microbial endosymbionts can affect the pathogenicity of Acanthamoeba. • This is the first report of some microbial endosymbionts in Acanthamoeba. • Further research is required to clarify the exact pattern of symbiosis, in order to modify treatment protocol. [ABSTRACT FROM AUTHOR]

Background: Free-living amoeba (FLA)-related keratitis is a progressive infection of the cornea with poor prognosis. The present study aimed to investigates the contact lenses of patients with keratitis for pathogenic free-living amoebae. Methods: Overall, 62 contact lenses and their paraphernalia of patients with keratitis cultured and tested for the presence of free-living amoebae using morphological criteria. Unusual plates including plates containing mix amoebae and Vermamoeba were submitted to molecular analysis. Results: Out of 62 plates, 11 revealed the outgrowth of free living amoeba of which 9 were Acanthamoeba, one plates contained mix amoebae including Acanthamoeba and Vermamoeba and one showed the presence of Vermamoeba. These two latter plates belonged to patients suffered from unilateral keratitis due to the misused of soft contact lenses. One of the patients had mix infection of Acanthamoeba (T4) and V. vermiformis meanwhile the other patient was infected with the V. vermiformis. Conclusion: Amoebic keratitis continues to rise in Iran and worldwide. To date, various genera of free-living amoebae such as Vermamoeba could be the causative agent of keratitis. Soft contact lens wearers are the most affected patients in the country, thus awareness of high-risk people for preventing free-living amoebae related keratitis is of utmost importance. [ABSTRACT FROM AUTHOR]